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PubMed1999

Growth factor signalling.

Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.

Animals↗
PubMed2001

Formation of unique vacuoles in tenotomized rat soleus muscle fibers.

The formation of unique vacuoles in tenotomized rat soleus muscle fibers was examined by light and electron microscopy. After tenotomy at both proximal and distal tendons, virtually all muscle fibers underwent characteristic degenerative changes with a disorganization of myofibrils called the central core lesion, but eventually recovered. At 3 days after tenotomy, some muscle fibers showed small vacuoles in the sarcoplasm of the end segments, which were larger in diameter and paler in staining than those of the control fibers in light microscopy. At 5 days, more fibers formed larger vacuoles together with the extensive disorganization of myofibrils. Such vacuole formation was more conspicuous in the distal end than in the proximal end. At 1 week the myofibrillar disorganization was most extensive in the central areas, and vacuoles were considerably enlarged in some fibers to occupy most of the sarcoplasm near the fiber ends. Vacuoles decreased in number and size with time and could rarely be seen at 4 weeks postoperative. In thin-section electron microscopy, the early forms of vacuoles were often connected with the T-system tubules. The limiting membrane of such vacuoles possessed many caveolae, some of which appeared to be continuous with the T-system networks. The vacuole membrane was closely associated with the sarcoplasmic reticulum to form dyadic connections. In later stages, the vacuole membrane was lined in part with the basal lamina. From these findings, it can be concluded that the vacuoles are sarcolemmal in nature and derived from the T-system. The significances of the vacuole formation are discussed with special reference to the mechanism and fate of the vacuoles and their clinical implications.

Animals↗
PubMed2005

Neural organization for recognition of grammatical and emotional facial expressions in deaf ASL signers and hearing nonsigners.

Recognition of emotional facial expressions is universal for all humans, but signed language users must also recognize certain non-affective facial expressions as linguistic markers. fMRI was used to investigate the neural systems underlying recognition of these functionally distinct expressions, comparing deaf ASL signers and hearing nonsigners. Within the superior temporal sulcus (STS), activation for emotional expressions was right lateralized for the hearing group and bilateral for the deaf group. In contrast, activation within STS for linguistic facial expressions was left lateralized only for signers and only when linguistic facial expressions co-occurred with verbs. Within the fusiform gyrus (FG), activation was left lateralized for ASL signers for both expression types, whereas activation was bilateral for both expression types for nonsigners. We propose that left lateralization in FG may be due to continuous analysis of local facial features during on-line sign language processing. The results indicate that function in part drives the lateralization of neural systems that process human facial expressions.

Adult↗
PubMed1992

Immunosuppression induced by hepatic portal venous immunization spares reactivity in IL-4 producing T lymphocytes.

Immunization of naive or specifically primed C3H/HEJ with irradiated B10.BR spleen cells via the hepatic portal vein leads to an antigen specific decrease in the proliferative and cytotoxic response to B10.BR antigen assayed in vitro (and to increased graft survival of B10.BR grafts in vivo). This effect seems to be mediated in the main by a decrease in IL-2 production from CD4+ T lymphocytes of mice given antigen by the portal route, which is in turn caused by a decreased precursor frequency of IL-2-producing cells. No clear decrease in IL-4 production was seen. Hepatic APC isolated from mice receiving antigen via the portal vein were unable to induce IL-2 production from a C3H/HEJ anti-B10.BR cell line in vitro, in contrast to splenic APC derived from the same mice. Even when antigen was given by conventional (systemic) intravenous routes (in this case via the lateral tail vein) hepatic APC isolated from those mice were unable to stimulate IL-2 production from this cell line. Furthermore, 24 h exposure of a cell line to antigen pulsed hepatic APC left those cells refractory to a subsequent restimulation with antigen presented by splenic APC. Spleen lymphoid cells from primed mice challenged in vivo with B10.BR liver cells (i.v.) were similarly unable to produce IL-2 on rechallenge in vitro with irradiated B10.BR spleen cells, though no defect was seen if in vivo challenge was with B10.BR spleen cells. These data imply that presentation of multiple minor cell surface antigens by hepatic APC leads to specific anergization of IL-2 producing T cells, in a fashion which seems to be distinct from that previously reported as due to 'veto-like' activity.

Animals↗
PubMed1988

A real-time data-logger system using an optical disk WORM for archiving continuous 12-lead ECG data during exercise testing.

An exercise ECG analysis program was developed over 15 years on a number of mainframes, minicomputers and, most recently, microcomputer-based systems. It has been rehosted into both Motorola MC68000 and Intel 80286 microprocessor-based development systems and is currently used with a removable 200 Mbyte optical disk (Write-Once-Read-Many, WORM) based data-logger system that can record and store all 12 leads simultaneously and continuously for an entire exercise test (up to 38 minutes). Data is acquired with 12-bit A/D resolution at 500 samples/sec. All ECG data and patient information are archived on the optical disk for later off-line recall and analysis on a PC or real-time replay through a D/A converter. Recorded ECG signals are at patient levels so they can be replayed through the patient cable box on any commercial system. Current development includes both simultaneous on-line processing and storage of 12-lead ECG data and off-line processing and development performed on the long-term, continuous ECG data being archived on optical disk. Patient medical histories and clinical information are separately entered into an applications database, where ECG measures and test results are later included. This new optical disk based exercise ECG database contains more than 600 complete exercise tests and is projected to increase to nearly 3,000 within 2 years.

Computer Systems↗
PubMed1999

The zebrafish eya1 gene and its expression pattern during embryogenesis.

The eyes absent-like genes encode a group of putative transcriptional coactivators with a sole representative in Drosophila and several members in mammals. Haploinsufficiency of the human EYA1 gene results in branchio-oto-renal syndrome characterized by developmental anomalies of the branchial arches, the three compartments of the ear and the kidney. As a first step towards a functional analysis of this gene in lower vertebrates, we isolated its zebrafish homologue, eya1, and studied its expression pattern during embryogenesis. The eya1 cDNA predicts a protein with 84.7% identity with the human homologue. Transcripts are first detected at the tailbud stage in presumptive cranial placodal precursor cells. Thereafter, eya1 expression continues in anterior pituitary, olfactory, otic, and lateral line placodes. Aside from these placodal sites of expression, eya1 transcripts were observed in the somites, developing pectoral fins, and branchial arches. No expression was found in pronephros or Wolffian duct of the zebrafish renal system. Within the developing ear, eya1 expression becomes confined to the ventral part of the otic vesicle from where the acoustic ganglion precursor cells arise and the sensory patches differentiate. In the lateral line, eya1 is expressed in the placodes, ganglia, migrating primordia, and receptive organs at all developmental stages, including both the differentiating hair and supporting cells. Taken together, these results indicate a remarkable similarity in both the structure and expression pattern of eya1 between higher and lower vertebrates, suggesting that the function of this gene has been conserved throughout vertebrate evolution.

Amino Acid Sequence↗
PubMed1997

Amyotrophic lateral sclerosis immunoglobulins increase intracellular calcium in a motoneuron cell line.

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca2+]i) in differentiated VSC4.1 cells. These transient [Ca2+]i increases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 microM) of omega-agatoxin IVa. The incidence of transient [Ca2+]i increases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca2+]i increases (addition of synthetic FTX or omega-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca2+]i increases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca2+]i changes induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis.

We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta 2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

Animals↗
PubMed2001

[Field matching in breast irradiation]

INTRODUCTION: In this paper the authors have combined different irradiation techniques for breast and adjacent supraclavicular lymph nodes. The aim was to reduce inhomogeneity in the match-line. METHODS: The CadPlan 6.1.5 three-dimensional treatment planning system was applied in this study for CT based plan using a standard medial and lateral wedged tangential breast portals with the adjacent supraclavicular field. Isocenter is placed at depth on the match-line, where asymmetric jaws are used to produce non-divergent field edges. The tangential fields are shaped using multi-leaf collimator (MLC), by following the curvature of the thorax. In this way the cranial vertical match plane is maintaned without using the breast board. The prescribed dose was 50 Gy at the isocentre. RESULTS: The calculated dose distributions were evaluated in three dimension in the match region of supraclavicular field and the two opposing tangential fields. This method produces a more uniform dose distribution in the target volume and in the match-line. Set-up is fast, this is done without the need for table rotation, or vertical cephalad blocks. The average dose to the ipsilateral lung is reduced using the IMRT (intensity modulated radiotherapy) technique by approximately 10% compared with the conventional technique. Furthermore, this new technique has the possibility to improve the field match between the tangential fields and the parasternal field, while maintaning the field match between the tangential fields and the axillary and supraclavicular fields.

Journal Article↗
PubMed1998

Morphology of the brain and sense organs in the snailfish Paraliparis devriesi: neural convergence and sensory compensation on the Antarctic shelf.

The Antarctic snailfish Paraliparis devriesi (Liparidae) is an epibenthic species, inhabiting depths of 500-650 m in McMurdo Sound. Liparids are the most speciose fish family in the Antarctic Region. We examine the gross morphology and histology of the sense organs and brain of P. devriesi and provide a phyletic perspective by comparing this morphology to that of four scorpaeniforms and of sympatric perciform notothenioids. The brain has numerous derived features, including well-developed olfactory lamellae with thick epithelia, large olfactory nerves and bulbs, and large telencephalic lobes. The retina contains only rods and exhibits a high convergence ratio (82:1). Optic nerves are small and nonpleated. The tectum is small. The corpus of the cerebellum is large, whereas the valvula is vestigial. The rhombencephalon and bulbospinal junction are extended and feature expanded vagal and spinal sensory lobes as well as hypertrophied dorsal horns and funiculi in the rostral spinal cord. The lower lobes of the pectoral fins have taste buds and expanded somatosensory innervation. Although the cephalic lateral line and anterior lateral line nerve are well developed, the trunk lateral line and posterior lateral line nerve are reduced. Near-field mechanoreception by trunk neuromasts may have been compromised by the watery, gelatinous subdermal extracellular matrix employed as a buoyancy mechanism. The expanded somatosensory input to the pectoral fin may compensate for the reduction in the trunk lateral line. The brains of P. devriesi and sympatric notothenioids share well-developed olfactory systems, an enlarged preoptic-hypophyseal axis, and subependymal expansions. Although the functional significance is unknown, the latter two features are correlated with habitation of the deep subzero waters of the Antarctic shelf.

Animals↗
PubMed2001

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.

Animals↗
PubMed1988

Serotonin and/or an excitatory amino acid in the medial medulla mediates stimulation-produced antinociception from the lateral hypothalamus in the rat.

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. Descending inhibition from the LH relies, at least in part, on a relay(s) in the midbrain and/or medulla. The medullary nucleus raphe magnus (NRM) serves as one such relay. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to systematically examine the transmitter(s) in the medial medulla mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of pharmacologic receptor antagonists (5 micrograms) into the NRM revealed that the glutamate receptor antagonists, gamma-D-glutamylglycine and 2-amino-5-phosphonovalerate produced the largest increases in stimulation thresholds in the LH for inhibition of the TF reflex (107.6% and 102.6%, respectively). Methysergide, a serotonin receptor antagonist, also produced a significant increase (81.5%) in the stimulation threshold in the LH for inhibition of the TF reflex. The opioid receptor antagonist, naloxone, however, was without effect, producing only a 4.0% increase in the LH stimulation threshold. These results suggest that serotonin and/or an excitatory amino acid are transmitters at the bulbar relay in the medial medulla mediating descending inhibition of the TF reflex produced by focal electrical stimulation in the LH.

Amino Acids↗
PubMed1999

Temporal and quantitative expression of the myelin-associated lipids, ethanolamine plasmalogen, galactocerebroside, and sulfatide, in the differentiating CG-4 glial cell line.

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [3H]ethanolamine to label ethanolamine plasmalogens or with [3H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.

Animals↗
PubMed1993

Determination of the center of pressure of the hoofs of the forelimbs of horses standing on a flat level surface.

The pressure exerted on a flat level surface by recently trimmed, unshod hoofs of the front limbs of 23 sound, adult horses was measured using pressure-sensitive film and a specially built cassette. The horses were tranquilized and stood with one foot on the 2.9-cm-thick cassette and the other on a block of equal height. The hoofs were observed for motion during the measurement, and the developed film was examined for improper alignment of the film or slipping of the hoof. The center of pressure was located using the method of weighted proportions of Barrey. This static measurement system with a long measurement time and the number of measurements reduced the influence of variables inherent in the horses' behavior and the measuring system. The calculated point was recorded as falling medial to, lateral to or on a line bisecting the central sulcus of the frog. In the dorsal to palmar orientation the point was classified with reference to a line drawn halfway between the most dorsal and the most palmar mark on the film. Forty-six percent of the calculated centers of pressure were located in the medial heel area. Binomial analysis for large samples indicates that this was a significant variation from a random distribution. Seventy-six percent of the centers were located in or on the borders of the medial heel.

Animals↗
PubMed1988

Brain-stem relays mediating stimulation-produced antinociception from the lateral hypothalamus in the rat.

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to examine systematically the organization in the brain stem of pathways mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of lidocaine into the midbrain, dorsolateral pons, or medial medulla resulted in significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (89.1, 67.4, and 73.6%, respectively). Selective lesions of cell bodies in the midbrain or medulla by the neurotoxin ibotenic acid also produced significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (31.6 and 131.6%, respectively), thus revealing relays in the periaqueductal gray and the nucleus raphe magnus located between the LH and the lumbar spinal cord. The failure of ibotenic acid to affect LH-produced descending inhibition when microinjected into the dorsolateral pons, and the significant effect produced by lidocaine microinjected into the same area, implicates fibers of passage in the dorsolateral pons in descending inhibition of the TF reflex produced by focal electrical stimulation in the LH. The fluorescent dye Fast blue and HRP conjugated to wheat germ agglutinin were used to confirm that the area stimulated in the LH has reciprocal connections with the periaqueductal gray and nucleus raphe magnus.

Animals↗
PubMed1985

Sensory neuron growth cones comigrate with posterior lateral line primordial cells in zebrafish.

The development of neuromasts and sensory neurons of the posterior lateral line was studied in zebrafish (Brachydanio rerio) in order to determine the relationship between growing axons of sensory neurons and the migratory cellular primordium of midbody line neuromasts. Scanning electron microscopy revealed that a primary system of six neuromasts develops during the second day after fertilization and evidence is presented that these arise from cells of a migratory primordium. The primordium is first detected in the postauditory region immediately adjacent to the developing sensory ganglion. Growth cones of posterior lateral line sensory neurons are found within the premigratory primordium when it is adjacent to the ganglion. At later times growth cones of these sensory neurons are found within the primordium as it migrates caudally along the midbody line. These results demonstrate that although the growth cones of the sensory neurons grow over a considerable distance to their final destination, they are never very far from their target cells (or target cell precursors), which migrate with them and may even lead them.

Animals↗